Melanocortins (a variety of different peptide products resulting from post-translational processing of pro-opiomelanocortin) are known to have a broad array of physiological actions. Aside from their well known effects on adrenal cortical function (e.g., by ACTH, adrenocorticotropic hormone), and on melanocytes (e.g., by .alpha.-MSH, melanocyte stimulating hormone), melanocortins have been shown to affect behavior, learning, and memory, control of the cardiovascular system, analgesia, thermoregulation, and the release of other neurohumoral agents including prolactin, luteinizing hormone, and biogenic amines. Peripherally, melanocortins have been identified to have immunomodulatory and neurotrophic properties and to be involved in events surrounding parturition.
The melanocortins mediate their effects through melanocortin receptors (MC-R)--a subfamily of G-protein coupled receptors. Other than the MC1-R which was identified as specific for .alpha.-MSH, and MC2-R which was identified as specific for ACTH, the melanocortin receptors cloned and identified to date (MC3-R, MC4-R, MC5-R) are thought of as "orphan" receptors--i.e., the identity of the native ligand for each receptor remains unidentified, and the physiologic function of each receptor type remains unknown.
The agouti protein is a gene product expressed in mice that is known to be involved in determining coat color, but also thought to play a role in obesity when its normal expression pattern is de-regulated and the protein is ubiquitously expressed. The receptor for agouti has not been identified or cloned; however, it has been observed that agouti antagonizes the MSH-induced activation of two melanocortin receptors.
2.1. THE MELANOCORTIN RECEPTORS
The first two melanocortin receptors cloned were the melanocyte MSH receptor, MC1-R, and the adrenocortical ACTH receptor, MC2-R (Mountjoy et al., 1992, Science 257:1248-1251; Chhajlani & Wikberg, 1992, FEBS Lett. 309:417-420). Subsequently, three additional melanocortin receptor genes were cloned which recognize the core heptapeptide sequence (MEHFRWG) of melanocortins. Two of these receptors have been shown to be expressed primarily in the brain, MC3-R (Roselli-Rehfuss et al., 1993, Proc. Natl. Acad. Sci. USA 90:8856-8860; Gantz et al., 1993, J. Biol. Chem. 268:8246-8250) and MC4-R (Gantz et al., 1993, J. Biol. Chem. 268:15174-15179; Mountjoy et al., 1994, Mol. Endo. 8:1298-1308). A fifth melanocortin receptor (originally called MC2-R) is expressed in numerous peripheral organs as well as the brain (Chhajlani et al., 1993, Biochem. Biophys. Res. Commun. 195:866-873; Gantz et al., 1994, Biochem. Biophs. Res. Commun. 200:1214-1220). The native ligands and functions of these latter three receptors remain unknown.
Because of their "orphan" status as receptors without an identified ligand, and the absence of any known physiological role for these new receptors, investigators have attempted to characterize the receptors in vitro, by their ability to bind and respond (e.g., transduce signal) to a variety of known melanocortins (e.g., see Roselli-Rehfuss, 1993, supra; and Gantz, 1993 supra) or agonists and antagonists derived from MSH and ACTH amino acid sequences (e.g., see Hruby et al., 1995, J. Med. Chem. 38:3454-3461; and Adan et al., 1994, Eur. J. Pharmacol. 269:331-337). In another approach, the members of the melanocortin receptor family were differentiated on the basis of their pattern of tissue distribution as a means for hypothesizing a function (e.g., See Gantz, 1993, supra; and Mountjoy 1994, supra). For example, expression of MC1-R is localized to melanocytes, MC2-R is localized to adrenal cortical cells, whereas the MC3-R and MC4-R are found primarily in the brain but not in the adrenal cortex or melanocytes; MC4-R is not expressed in the placenta, a tissue that expresses large amounts of MC3-R. Based upon its expression pattern in the hippocampal region of the brain, a role for the MC4-R in learning and memory was proposed (Gantz, 1993, supra) but was noted to be a "pharmacological paradox" in that the MC4-R does not respond well to compounds known to have an effect on retention of learned behaviors. (Mountjoy, 1994, supra). Mountjoy 1994 further suggests that the MC4-R may participate in modulating the flow of visual and sensory information, or coordinate aspects of somatomotor control, and/or may participate in the modulation of autonomic outflow to the heart.
Thus, despite such efforts, the native ligands and function of MC3-R, MC4-R and MC5-R remain elusive.
2.2. THE AGOUTI PROTEIN
The agouti gene is predicted to encode a secreted protein expressed in hair follicles and the epidermis, the expression of which correlates with the synthesis of the yellow pigment associated with the agouti phenotype (Miller et al., 1993, Gene & Development 7:454-467). Certain dominant mutations of the agouti gene result in de-regulated, ubiquitous expression of the agouti protein in mice, demonstrating pleiotropic effects that include obesity and increased tumor susceptibility. (Miller et al., 1993, supra; Michaud et al., 1993, Genes & Development 7:1203-1213). Ectopic expression of the normal, wild-type, agouti protein in transgenic mice results in obesity, diabetes, and the yellow coat color commonly observed in spontaneous obese mutants (Klebig, et al., 1995, Proc. Natl. Acad. Sci. USA 92:4728-4732). For reviews, see Jackson, 1993, Nature 362:587-588; Conklin & Bourne, 1993, Nature 364:110; Siracusa 1994, TIG 10:423-428; Yen et al., 1994, FASEB J. 8:479-488; Ezzell, 1994, J. NIH Res. 6:31-33; and Manne et al., 1995, Proc. Sci. USA 92:4721-4724.
No receptor for agouti has been identified. Agouti has been reported to be a competitive antagonist of AMSH binding to the MC1-R and MC4-R in vitro (Lu et al., 1994, Nature 371:799-802), and the authors speculated that ectopic expression of agouti may lead to obesity by antagonism of melanocortin receptors expressed outside the hair follicle. In this regard, a number of theories have been proposed to account for the induction of obesity by ectopic expression of agouti. For example, agouti expression in skeletal muscle may result in insulin resistance, hyperinsulinemia and obesity via elevation of Ca.sup.2 +levels; alternatively ectopic agouti expression in adipocytes may depress lipolysis; conversely direct effects of agouti on pancreatic .beta. islet cells may result in hyperinsulinemia and obesity; yet another possibility is that agouti expression in the brain may result in obesity due to a primary effect on areas of the brain controlling weight regulation and insulin production (see Klebig 1995, supra).
In sum, the mechanism of agouti-induced obesity in mice is unknown, and the relevance, if any, of this phenomenon to human obese phenotypes has not been established.